Báo cáo khoa học: The variable C-terminal extension of G-protein-coupled receptor kinase 6 constitutes an accessorial autoregulatory domain ppt

The variable C-terminal extension of G-protein-coupled receptor kinase constitutes an accessorial autoregulatory domain Petra Vatter*, Claudia Stoesser*, Ines Samel, Peter Gierschik and Barbara Moepps Department of Pharmacology and Toxicology, University of Ulm, Germany Keywords desensitization; G-protein-coupled receptor kinases; G-protein-coupled receptors; phosphorylation; signal transduction Correspondence B Moepps, Department of Pharmacology and Toxicology, University of Ulm, AlbertEinstein-Allee 11, 89081 Ulm, Germany Fax: +49 731 5002 3872 Tel: +49 731 5002 3883 E-mail: barbara.moepps@uni-ulm.de *Petra Vatter and Claudia Stoesser contributed equally to this work (Received August 2005, revised 17 September 2005, accepted 27 September 2005) doi:10.1111/j.1742-4658.2005.04995.x G-protein-coupled receptor kinases (GRK) are known to phosphorylate agonist-occupied G-protein-coupled receptors We expressed and functionally characterized mouse GRK6 proteins encoded by four distinct mRNAs generated by alternative RNA splicing from a single gene, mGRK6-A to mGRK6-D Three isoforms, mGRK6-A to mGRK6-C differ in their C-terminal-most portion, which is known to mediate membrane and ⁄ or receptor interaction and regulate the activity of GRK4-like kinases One isoform, mGRK6-D, is identical to the other mGRK6 variants in the N-terminal region, but carries an incomplete catalytical domain Mouse GRK6-D was catalytically inactive and specifically present in the nucleus of transfected cells Recombinant mouse GRK6-A to mGRK6-C were found to be membrane-associated in cell-free systems and in transfected COS-7 cells, suggesting that the very C-terminus of GRK6-A, lacking in GRK6-B and mGRK6-C and carrying consensus sites for palmitoylation, is not required for membrane interaction Interestingly, the shortest catalytically active variant, mGRK6-C, was conspicuously more active in phosphorylating light-activated rhodopsin than mGRK6-A and mGRK6-B, implying that the C-terminus of the latter two variants may fulfil an autoinhibitory function Mutation and removal of C-terminal-most region of mGRK6-A by site-directed mutagenesis revealed that this region contains three autoregulatory elements: two discontinuous inhibitory elements consisting of a single residue, D560, and the sequence between residues S566 and L576, and an intervening stimulatory element The results suggest that mGRK6C may be considered a basic, prototypic representative of the GRK4-like kinases, which is capable of interacting with both plasma membrane and its receptor substrate, but is resistant to further regulatory modification conferred to the prototype via C-terminal extension Phosphorylation by members of a family of serine– threonine protein kinases, termed G-protein-coupled receptor kinases (GRKs), has been shown to be crucial in the rapid agonist-induced desensitization of many G-protein-coupled receptors [1–4] Seven members of the mammalian GRK family (GRK1–7), each encoded by a single gene, have been described Diversity of this protein family is further enhanced by alternative RNA splicing, as shown for human GRK4 [5], human rhodopsin kinase (GRK1) [6], and murine and human Abbreviations CaM kinase, calmodulin-dependent protein kinase; G protein, heterotrimeric guanine nucleotide binding protein; GRK, G protein-coupled receptor kinase; PH, pleckstrin homology; PKC, protein kinase C; PtdCho, phosphatidylcholine; PtdIns, phosphatidylinositol; PtdInsP, phosphatidylinositol 4-phosphate; PtdInsP2, phosphatidylinositol 4,5-bisphosphate; PtdSer, phosphatidylserine; Sf9, Spodoptera frugiperda Spodoptera frugiperda FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS 6039 Variable C-terminus of GRK6 mediates autoregulation P Vatter et al GRK6 [7,8] Based on similarities in their primary structures and functional properties, GRKs are grouped into three subfamilies [9] GRK1 and GRK7 are members of the first subfamily, GRK2 and GRK3 compose the second subfamily, and GRK4, GRK5 and GRK6 make up the third subfamily, also referred to as the GRK4 subfamily GRKs share a similar structural organization characterized by a centrally located, highly homologous catalytical domain flanked by variable N- and C-terminal regions [1,2] The N-terminal regions of the known mammalian GRKs are similar in size and have been proposed to be involved in receptor recognition [10] The C-terminal regions of the known mammalian GRKs, which share a certain degree of conservation, but vary in length [11], are generally thought to play an important role in localizing the kinases to the plasma membrane [2,12] This region can be further subdivided into two constituents: a moderately conserved N-terminal region consisting of  65 residues (residues 449–514 in mGRK6-A to mGRK6-C) [7] and a highly variable C-terminal region [13] The variable region contains sites for regulatory protein–protein interactions and ⁄ or post-translational modification, e.g isoprenylation or palmitoylation, which are thought to be involved in targeting of GRKs to the receptor and ⁄ or the plasma membrane We have previously identified mRNAs encoding four distinct mouse GRK6 isoforms (mGRK6), designated mGRK6-A to -D [7] Analysis of the genomic organization of the mouse GRK6 gene showed that the four mRNAs are generated by alternative RNA splicing from a single gene Three of the GRK6 isoforms, GRK6-A, -B and -C, are likely to exist in human and rat tissues as well [8] (P Vatter, C Stoesser, I Samel, P Gierschik & B Moepps, unpublished results) Expression studies revealed that the four mGRK6 mRNAs are differentially expressed in mouse tissues, suggesting that the four mGRK6 isoforms are involved in regulating tissue- or cell-type-specific receptor functions in vivo [7] Interestingly, mGRK6-A, but not mGRK6-B or -C, phosphorylated the Na+ ⁄ H+ exchanger regulatory factor (NHERF) via a PDZdomain-mediated interaction, presumably mediated by the PTRL motif specifically present at the C-terminus of this GRK6 isoform [14] The gene encoding GRK6 has been inactivated in the mouse [15] Interestingly, lymphocytes derived from GRK6-deficient mice were strikingly impaired in their ability to respond to the CXC chemokine CXCL12, indicating that GRK6 is specifically involved in regulating the CXCL12 receptor CXCR4 [15,16] In addition, dopamine D2 receptors and leukotriene B4 receptors have been shown to 6040 be regulated by GRK6 [17,18] The GRK6 variants mGRK6-A, -B and -C are indistinguishable up to residue 559 of the variable C-terminal region and diverge from each other at residue 560 Mouse GRK6-A is identical to human GRK6-A between residues 560 and 576 and therefore contains the three cysteine residues known to undergo palmitoylation in hGRK6-A within its C-terminus [19,20] The C-terminus of mGRK6-B is 13 residues longer than the corresponding portion of mGRK6-A and lacks these palmitoylation sites, but carries several basic residues and consensus sequences for phosphorylation by protein kinase C and cAMP ⁄ cGMP-dependent protein kinases The unique C-terminus of mGRK6-C is made up of only a single arginine residue present in position 560 In this study, we expressed the four variants of mouse GRK6 as recombinant polypeptides in both baculovirus-infected insect and in COS-7 cells to characterize the functional significance of their structural differences The results show that mGRK6-D is catalytically inactive and specifically present in the nucleus of transfected COS-7 cells In contrast, all three forms carrying a complete catalytical domain are catalytically active and localized to the plasma membrane in intact cells The observation that mGRK6-C, lacking the C-terminal extensions present in mGRK6-A and -B and likely to serve as substrates for palmitoylation or protein phosphorylation, respectively, is considerably more active than the latter two variants, suggests that the C-terminal extensions are not required for membrane interaction Instead, they appear to fulfil an accessorial autoregulatory function subject to further, reversible modification by palmitoylation or protein phosphorylation, respectively Results Production of polyclonal antisera against mouse GRK6 variants To characterize the four mouse GRK6 variants at the protein level, the polypeptides encoded by the four mGRK6 mRNAs were expressed in Sf9 insect cells and COS-7 cells To monitor expression and to examine the subcellular distribution of the recombinant proteins, two polyclonal antisera reactive against the various mGRK6 isoforms were produced Serum PV1 was raised against a peptide located in the N-terminal portion present in all four isoforms (amino acids 87– 101) Serum PV2 was directed against a peptide from the unique C-terminus of mGRK6-B (amino acids 574–589) To specifically detect mGRK6-A, a commercially available polyclonal antiserum (sc-566) reactive FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS P Vatter et al against the C-terminus mGRK6-A (amino acids 557– 576) was used (cf Fig 1A) To determine the specificity of the antisera, all four mGRK6 variants were expressed as recombinant polypeptides in baculovirus-infected insect cells and analysed by immunoblotting Figure 1B shows that all four polypeptides migrated at the expected positions on SDS polyacrylamide gels and were reactive with serum PV1 (left) In contrast, mGRK6-A (centre) and mGRK6-B (right) were specifically detected by antisera sc-566 and PV2, respectively No immunoreactivity was seen using antisera PV1, PV2 or sc-566 in noninfected insect cells A Variable C-terminus of GRK6 mediates autoregulation (results not shown) Using antisera sc-566 and PV2 to detect mGRK6-A and -B in membrane preparations of mouse tissues by immunoblotting (results not shown), we found that mGRK6-A was expressed in brain, liver, heart and spleen and, at lower levels, in lung, mesenterial lymph nodes and thymus Mouse GRK6-B was present at high levels in brain, followed by spleen, mesenterial lymph nodes and thymus, but was undetectable in liver and heart Attempts at specifically detecting mGRK6-C on immunoblots were unsuccessful due to the fact that this variant differs from mGRK6-A and -B in but a single residue (R560, cf Fig 7A), and that it was not possible to resolve the three variants by SDS ⁄ PAGE Nevertheless, these results clearly indicate that the splice variants of mGRK6 are differentially expressed at the protein level in mouse tissues Subcellular localization of the mouse GRK6 variants in transfected COS-7 cells B Fig Expression of four mouse GRK6 proteins in baculovirusinfected insect cells (A) Linear representation of the four mGRK6 variants generated by alternative RNA splicing The predicted catalytic domain and the regulatory N- and C-terminal regions are shown as shaded and open boxes, respectively The variable C-terminal regions of mGRK6-A, -B and -C are shown in detail The residues predicted to serve as substrates for palmitoylation in mGRK6-A and for phosphorylation by protein kinases C and cAMP ⁄ cGMP-dependent protein kinases in mGRK6-B, respectively, are underlined Mouse GRK6-D terminates at residue 244 within the predicted catalytic domain (dashed vertical line) The positions of the synthetic peptides used to generate antisera PV1, sc-566 and PV2 are illustrated The numbering of the amino acid residues is indicated (B) Sf9 cells were infected with baculoviruses encoding the mGRK6 variants mGRK6-A, -B, -C and -D Forty-eight hours after infection, cells were homogenized with lysis buffer containing 250 mM NaCl and the homogenate was fractionated into soluble and particulate constituents Aliquots of the soluble fractions of insect cells expressing mGRK6-A, -B, -C and -D containing 9, 13, and 45 lg of protein, respectively, were subjected to SDS ⁄ PAGE and immunoblotting was performed using antisera PV1 (left), sc-566 (centre) or PV2 (right) Soluble proteins from noninfected Sf9 cells (Sf9) (7 lgỈlane)1) were analysed for comparison The positions of the molecular mass standards are indicated To examine the subcellular distribution of the recombinant mGRK6 variants in cultured mammalian cells, COS-7 cells were transiently transfected with cDNAs encoding mGRK6-A to -D Transfected cells were homogenized in low ionic strength buffer, unbroken cells and nuclei were removed from the homogenate, and the postnuclear supernatant was separated into soluble and particular components In addition, the particulate material was extracted with buffer containing Triton X-100 (1.5% w ⁄ v) Figure 2A shows the results of an immunochemical analysis of the soluble and particular fractions as well as the detergent extracts of these samples Recombinant mGRK6-A and -B were exclusively, and mGRK6-C predominantly present in the particulate fraction of transfected cells Thus, only a small fraction of mGRK6-C appeared in the soluble fraction under the conditions used in this experiment All three variants were solubilized from the particulate fraction with buffer containing Triton X-100 No immunoreactivity was found for mGRK6-D in soluble or particulate postnuclear fractions of COS-7 cells transfected with the mGRK6-D cDNA (not shown) Next, the subcellular localization of the mGRK6 isoforms was examined by immunocytochemistry and confocal microscopy using antiserum PV1 and fluorescent-labelled secondary antibodies Figure 2B shows that immunoreactivity corresponding to mGRK6-A, -B and -C was located at the cell membrane and, although to a lesser extend, in the cytoplasma Most interestingly, cells transfected with the cDNA of mGRK6-D displayed immunoreactive protein exclusively in regions FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS 6041 Variable C-terminus of GRK6 mediates autoregulation P Vatter et al A B Fig Expression of four mouse GRK6 proteins in transiently transfected COS-7 cells COS-7 cells were transfected as indicated with lg each per well of pMT2 containing the cDNAs of mGRK6-A, -B, -C or -D (A) Forty-eight hours after transfection, cells were homogenized and the homogenate was fractionated into soluble (S) and particular (P) constituents A portion of the particular fraction was extracted with buffer containing Triton X-100 to obtain a detergent-soluble lysate (D) Aliquots (D, S: 70 lg protein per lane; P: 40 lg protein per lane) of the samples were subjected to SDS ⁄ PAGE and immunoblotting was performed using antiserum PV1 (B) Forty-eight hours after transfection, the cells were fixed and permeabilized, and immunostaining of the mGRK6 variants was performed using antiserum PV1 Plasma membranes (pm) and nuclei (n) of cells expressing mGRK6-A, -B, -C or -D, respectively, are marked by arrows No immunostaining was observed in nontransfected COS-7 cells (not shown) corresponding to the cell nucleus Thus, mGRK6-D is expressed in transiently transfected COS-7 cells, but localized to the nuclear fraction, which explains the absence of the protein from the postnuclear fractions analysed in Fig 2A Untransfected COS-7 cells and mGRK6 cDNA-transfected cells incubated with the primary or secondary antibody alone displayed no immunoreactivity (not shown) Taken together, these results revealed that: (a) mGRK6-A, -B and -C appear to be at least partially cell membrane-associated in intact mammalian cells; and (b) mGRK6-D is specifically present in the nucleus of transfected COS-7 cells Because both mGRK6-B and mGRK6-C lack the putative C-terminal palmitoylation sites present in mGRK6-A and thought to mediate membrane binding, these data imply further sites and mechanisms to be important for membrane association of former two mGRK6 variants Interaction of mGRK6-C with phospholipids To study the interaction of recombinant mGRK6-C with lipid membranes in more detail, insect cells infected with baculovirus encoding mGRK6-C were homogenized in buffer containing increasing concentrations 6042 of NaCl and the postnuclear homogenate was separated into soluble and particulate constituents Figure shows that mGRK6-C was predominantly present in the particulate fraction in the absence of NaCl, but was translocated to a considerable extent into the soluble fraction at increasing concentrations of NaCl Thus, membrane binding of mGRK6-C is dependent on and inversely related to the ionic strength of the incubation medium Previous studies have suggested an important role of N- and C-terminal portions of members of the GRK4 subfamily in mediating their interaction with phospholipids, including phosphatidylinositol 4,5-bisphosphate (PtdInsP2) [21,22] To assess the potential role of these putative phospholipid-binding sites for targeting mGRK6-C to lipid membranes, we investigated the interaction of recombinant mGRK6-C to synthetic phospholipid vesicles made up of phosphatidylcholine (PtdCho) and a small fraction of either phosphatidylserine (PtdSer) or of the inositol phospholipids PtdIns, PtdInsP or PtdInsP2 To this end, mGRK6-C was expressed in baculovirus-infected insect cells and purified from the soluble fraction by sequential cation exchange and heparin-affinity chromatography Active mGRK6-C was assayed by FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS P Vatter et al Variable C-terminus of GRK6 mediates autoregulation Fig Effect of NaCl on the interaction of recombinant mGRK6-C with the particulate fraction of baculovirus-infecetd insect cells Sf9 cells were infected with baculovirus encoding mGRK6-C Forty-eight hours after infection, cells were homogenized with lysis buffer containing increasing concentrations of NaCl and the homogenate was fractionated into soluble (S) and particulate (P) constituents Aliquots (40 lg protein per lane) of the fractions were subjected to SDS ⁄ PAGE and immunoblotting was performed using antiserum PV1 Only the  65 kDa region of the chemiluminogram is shown C A B Fig Purification of mGRK6-C from Sf9 cells Recombinant mGRK6 was purified from the soluble fraction of baculovirus-infected insect cells by sequential chromatography on SP Sepharose and heparin Sepharose (A) Aliquots (5 lL) of the indicated fractions obtained by chromatography on heparin Sepharose were subjected to SDS ⁄ PAGE and immunoblotting using antiserum PV1 (B) Aliquots of the same fractions (1 lL) were incubated with urea-treated rod outer segment membranes and [32P]ATP[cP] to measure phosphorylation of light-activated rhodopsin Samples were subjected to SDS ⁄ PAGE and autoradiography was performed (C) An aliquot (4 lg) of fraction was subjected to SDS ⁄ PAGE and proteins were stained with silver The positions of the molecular mass standards are indicated Aliquots of the sample applied to the heparin Sepharose matrix (15 and lL, respectively) were analysed for comparison (Co) immunoblotting (Fig 4A) and by measuring the ability of the protein to phosphorylate light-activated rhodopsin (Fig 4B) Figure 4C shows that the protein was mostly homogenous after heparin-affinity chromatography The interaction of purified mGRK6-C with phospholipids was investigated by incubating the protein with increasing concentrations of phospholipid vesicles and then separating vesicle-bound from soluble mGRK6-C by ultracentrifugation Figure 5A shows that mGRK6-C did not interact with vesicles made up of PtdCho even at the highest concentration tested (10 lgỈmL)1) When PtdSer was present in the vesicles, only a minor portion of mGRK6-C sedimented with the particulate fraction In contrast, interaction of mGRK6-C was clearly evident already at low phospholipid concentrations when PtdInsP2 was present in the vesicles At the highest phospholipid concentration tested, all of the mGRK6-C protein was found in the vesicle fraction To examine the specificity of the effect of PtdInsP2 shown in Fig 5A, purified mGRK6-C was incubated with increasing concentrations of PtdCho vesicles containing PtdIns, PtdInsP or PtdInsP2 As shown in Fig 5B, all three inositol phospholipids promoted the interaction of mGRK6-C with the vesicle preparation However, the strength of this interaction was markedly dependent on the nature of inositol phospholipid Specifically, maximal phospholipid binding was observed in the presence of PtdInsP2, followed by PtdInsP and PtdIns FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS 6043 m G R K6 -B G m G m R K6 -C P Vatter et al R K6 -A Variable C-terminus of GRK6 mediates autoregulation Rho* Fig Phosphorylation of light-activated rhodopsin by mGRK6 variants Sf9 cells were infected with baculovirus encoding mGRK6-A, -B and -C Aliquots of the soluble fractions of infected cells containing similar amounts of recombinant mGRK6 proteins were subjected to SDS ⁄ PAGE and immunoblotting using antiserum PV1 (upper) or incubated with urea-treated rod outer segment membranes and [32P]ATP[cP] to measure phosphorylation of light-activated rhodopsin (Rho*) The samples were subjected to SDS ⁄ PAGE and autoradiography of the gel was performed (lower) The position of Rho* is indicated Fig Interaction of mGRK6-C with phospholipids Recombinant mGRK6 was purified to homogeneity from the soluble fraction of baculovirus-infected insect cells Aliquots (2 lg protein per sample) were incubated with increasing concentrations of phospholipid vesicles made up of either 100% phosphatidylcholine (PtdCho) or 95% (w ⁄ w) phosphatidylcholine and 5% (w ⁄ w) of the indicated phospholipids The incubation mixtures (30 lL) were fractionated into soluble (S) and particulate (P) constituents by centrifugation The soluble supernantant was removed and the membrane fraction was resuspended in 30 lL of incubation buffer Aliquots (15 lL per lane) were subjected to SDS ⁄ PAGE and immunoblotting was performed using antiserum PV1 Only the  65 kDa regions of the chemiluminograms are shown Functional properties of the mouse GRK6 isoforms The next experiment was designed to examine and compare the functional properties of the mGRK6 isoforms mGRK6-A, -B and -C The three recombinant proteins were produced in baculovirus-infected insect cells, adjusted to similar levels by immunoblotting, and then assayed for their ability to phosphorylate lightactivated rhodopsin (Fig 6) None of the variants phosphorylated rhodopsin in the dark (not shown) All three isoforms were capable of specifically phosphorylating the active receptor protein (Fig 6), whereas insect cell-expressed mGRK6-D was inactive in this regard (not shown) Most interestingly, the activity of mGRK6-C was by far higher than the activities of the 6044 C-terminally extended variants mGRK6-A and -B (Fig 6) These results suggested that the C-terminal extensions present in the latter two isoforms may reduce their ability to phosphorylate activated receptor polypeptides Structure–activity relationships of the C-terminus of mGRK6 To examine the functional significance of the C-terminus of mGRK6-A, a mutant was generated (mGRK6-A M1) carrying serine residues instead of the cysteine residues C561, C562 and C565, which are known to be substrates for palmitoylation in wild-type mGRK6-A Furthermore, deletion mutants of mGRK6-A M1 lacking the C-terminal-most (mGRK6-A M2) and 16 (mGRK6-A M3) residues were produced The rationale behind constructing these mutants was based on the desire to determine the influence of the C-terminus of mGRK6-A without the influence of its palmitoylation (Fig 7A) Wild-type mGRK6-A and -C as well as the three mutants of mGRK6-A M1, M2 and M3, were produced as recombinant proteins in baculovirus-infected insect cells The amounts of the recombinant proteins were adjusted to similar levels by immunoblotting and the proteins were assayed for their ability to phosphorylate lightactivated rhodopsin (Fig 7B) Replacement of the three FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS P Vatter et al A B Variable C-terminus of GRK6 mediates autoregulation mutant differs from wild-type mGRK6-C in only a single residue, D560 instead of R560 Thus, the very C-terminus of mGRK6-A contains at least three regulatory elements capable of markedly and specifically affecting the activity of the kinase towards the receptor substrate, which are shown schematically in Fig 7A: two discontinuous inhibitory elements consisting of a single residue, D560, and the sequence between residues S566 and L576, and an intervening stimulatory element Discussion Fig Effect of C-terminal deletions on mGRK6-A activity (A) Linear representation of the C-terminal regions of wild-type mGRK6-A and mGRK6-C, and of the mutants of mGRK6-A The residues predicted to serve as substrates for palmitoylation in wild-type mGRK6-A are underlined The positions of the regions suggested to be involved in membrane interaction containing a high number of basic residues and those suggested to be involved in negative (circled minus symbols) and positive (circled plus symbol) autoregulation are indicated The numbering of the amino acid residues is indicated (B) Sf9 cells were infected as indicated with baculovirus encoding either wild-type mGRK6-A or -C, or deletion mutants of mGRK6-A Aliquots of the soluble fractions of infected cells containing similar amounts of recombinant mGRK6 proteins were subjected to SDS ⁄ PAGE and immunoblotting using antiserum PV1 (upper) or incubated with urea-treated rod outer segment membranes and [32P]ATP[cP] to measure phosphorylation of light-activated rhodopsin (Rho*) The samples were subjected to SDS ⁄ PAGE and autoradiography of the gel was performed (lower) The position of Rho* is indicated C-terminal cysteines by serine residues resulted in an almost complete loss of the ability of the protein to phosphorylate light-activated rhodopsin Interestingly, gradual removal of the C-terminal-most 16 residues of mGRK6-A M1 affected the activity of the protein toward rhodopsin in a nonuniform manner Thus, removal of the C-terminal-most nine residues of mGRK6-A M1, S568 to L578, in the mutant mGRK6A M2 led to a marked increase in rhodopsin phosphorylation Further removal of seven residues, S561 to D567, in mutant mGRK6-A M3 caused a marked loss of this activity (Fig 7B, lower) Note that the latter We expressed the four variants of mouse GRK6 as recombinant polypeptides to characterize the functional significance of the differences in their primary structures Whereas mGRK6-A, -B and -C are similar in terms of their overall structural organization to the other members of the GRK family, mGRK6-D is peculiar in that it terminates prematurely in its putative catalytic domain [7] The finding reported here that mGRK6-D is catalytically inactive when overexpressed as recombinant protein in baculovirus-infected insect cells is consistent with this structural deficiency Interestingly, mGRK6-D, unlike the other variants of mGRK6, was specifically present in the nucleus of transiently transfected COS-7 cells Differential subcellular localization of protein products of alternatively spliced RNAs is not without precedence in the literature Thus, localization in different subcellular compartments including the nucleus has been shown for splice variants of the fibroblast growth factor receptor FGFR-3 [23] and multifunctional Ca2+ ⁄ calmodulindependent protein kinase (CaM kinase) [24,25] The CaM kinase splice variants dB-CaM and aB-CaM both carry an 11 amino acid insertion in their variable regions This insertion generates a nuclear localization signal, KKRK [26], which targets the proteins to the nuclei of transfected cardiac myocytes or neuroblastoma cells, respectively Interstingly, a KKRK motif is also present in the C-terminal region of mGRK6-D If this peptide does, in fact, represent a nuclear localization signal of mGRK6-D, it is likely to be inactive in the other three variants, because those variants also carry the motif, but are absent from the nucleus in transfected COS-7 cells (Fig 2B) As the KKRK motif is part of an intact catalytic domain in mGRK6-A, -B and -C, it is conceivable that this motif is inaccessible in those variants, but freely available in mGRK6-D to interact with the nuclear import receptor subunit importin a [27] The function of mGRK6-D in the nucleus is currently unknown The C-terminal-most portion of GRKs is thought to play an important role in mediating receptor and ⁄ or FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS 6045 Variable C-terminus of GRK6 mediates autoregulation P Vatter et al plasma membrane interaction Thus, the primary structures of GRK1 and GRK7 terminate with a CAAX sequence that directs post-translational isoprenylation, proteolysis, and carboxy-methylation [28] GRK2 and GRK3 are localized in the cytosol and are translocated to the plasma membrane upon stimulation of G-protein-coupled receptors [29] The translocation of GRK2 to the membrane has been shown to be due to the binding of a C-terminal region including a pleckstrin homology domain to membrane-bound G protein bc dimers and negatively charged membrane phospholipids, including PtdInsP2 [30–34] Mutations in the GRK2 pleckstrin homology domain and the region distal to the C-terminal amphipathic helix resulted in a specific and profound loss of GRK2 responsiveness to bc dimers and phospholipids [35] Members of the GRK4 subfamily, GRK4, GRK5 and GRK6, contain neither a CAAX motif nor a G protein bc dimer-binding domain in the variable region of the C-terminal region, but nevertheless exhibit a significant degree of association with cellular membranes [5,19,36] GRK4 and human GRK6-A are palmitoylated at cysteine residues present within their C-terminal regions, whereas GRK5 contains a C-terminal polybasic domain likely to mediate direct interaction with negatively charged phospholipid head groups [37] The fact that GRK6-C lacks the C-terminal cysteine residues likely to serve as substrates for palmitoylation in GRK6-A, together with the observation that the activity of GRK6-A is substantially increased by palmitoylation, led Premont et al [8] to suggest that GRK6-C may associate with membranes only poorly and may represent a poor regulator of G-proteincoupled receptors However, the degree of association of recombinant mGRK6-C with the plasma membrane lipid bilayer in transfected COS-7 cells was similar to that of mGRK6-A and mGRK6-B (Fig 2A) These results imply that the C-terminal-most portions of mGRK6-A and mGRK6-B are not necessary for membrane association and that other mechanisms may be involved in targeting mGRK6 to the plasma membrane Of interest, at least two phospholipid binding sites appear to be present in GRK5 to mediate interaction of this member of the GRK4-like kinases with the plasma membrane [21,22] The first site, K22RKGKSKK in bovine GRK5 (basic residues underlined), is located at the N-terminus of GRK5 and appears to specifically interact with PtdInsP2 via its basic residues Binding of PtdInsP2 to this site markedly enhances GRK5-mediated phosphorylation of the human b2-adrenoceptor, most likely by facilitating the interaction of the kinase with the lipid bilayer [21] The second, C-terminally located site, 6046 Q552RLFKRQHQNN in human GRK5 (basic residues underlined), is critical for the interaction of GRK5 with artificial phospholipid vesicles in cell-free systems and with the plasma membrane in intact cells [22] Importantly, similar sites are present in GRK4 and in GRK6-A to -C Specifically, the N-terminal site corresponds to K21QTGRSKK in mGRK4 and to N22RKGKSKK in mGRK6-A to -C The C-terminal site corresponds to R552RLFRRTGCLN in mGRK4, to Q553RLFSRQDCCG in mGRK6-A, to Q553RL FSRQRIAV in mGRK6-B, and to Q553RLFSRQR in mGRK6-C Note that an acidic residue, D560, is present in the C-terminal motif of mGRK6-A in place of the basic residue, R560, present in mGRK6-B and -C, which may reduce the electrostatic potential of the former motif to interact with phospholipids In work to be published elsewhere (C Stoesser et al., unpublished results), we found that both the N-terminal and the C-terminal phospholipid-binding sites of mGRK6-C are involved in the interaction of this mGRK6 variant with phospholipid vesicles containing PtdInsP2 These observations suggest that two distinct phospholipidsbinding sites commonly present in mGRK6-A to -C mediate or contribute to the interaction of these mGRK6 variants with the plasma membrane Previous studies have shown that the C-terminal regions of GRK4 subfamily members are involved not only in membrane targeting, but also in regulation of substrate recognition and ⁄ or catalytic activity Thus, palmitoylation and artificial geranyl geranylation of the C-terminus of human GRK6-A enhanced phosphorylation not only of the human b2-adrenoceptor reconstituted into phospholipid vesicles, but also of the soluble nonreceptor substrate casein [38] These results strongly suggest that these post-translational modifications not only enhance the hydrophobicity and thereby strengthen the membrane association of GRK6-A, but also increase the kinase catalytic activity of the protein Along the same lines, the C-terminal 28 residues immediately downstream of the C-terminal phospholipid-binding site of human GRK5 have previously been suggested to fulfil an autoinhibitory function, which is enhanced by protein kinase C-mediated phosphorylation and ⁄ or autophosphorylation [22] Thus, phosphorylation of two or three serine residues within this region by protein kinase C dramatically reduced the ability of the kinase to phosphorylate both receptor and soluble nonreceptor substrates without affecting GRK5 binding to artificial phospholipid vesicles [39] The same region also contains three serine residues, which are potential sites for Ca2+-calmodulinstimulated autophosphorylation of human GRK5 [22] Phosphorylation of one or several of these residues FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS P Vatter et al Variable C-terminus of GRK6 mediates autoregulation causes substantial inhibition of the interaction of GRK5 with the receptor substrate without disrupting the catalytic activity or the association of GRK5 with phospholipids [40] The results reported in this study indicate that the C-terminal-most 16 residues of mGRK6-A may function as an autoregulatory domain to control the activity of this kinase towards the receptor substrate Inhibitory effects of this region are most clearly evident for the first residue, D560, and the last nine residues that discriminate mGRK6-A from mGRK6-C, S568EEELPTRL576 The seven residues between positions 561 and 567, which comprise the positions of the cysteine residues serving as substrates for palmitoylation in wild-type mGRK6-A (C561, C562 and C565) appear to exert a stimulatory effect It is tempting to speculate that palmitoylation of one or several of these cysteines may further enhance this stimulatory effect Taken together, we have shown that all three isoforms of mGRK6 containing a complete catalytic domain, mGRK6-A, mGRK6-B and mGRK6-C, are localized to the plasma membrane in intact cells The C-terminal extensions that discriminate mGRK6-A and -B from mGRK6-C are not required for membrane interaction, but instead appear to fulfil an accessorial autoregulatory function subject to reversible modification by palmitoylation or protein phosphorylation, respectively The fact that GRK6-A and GRK6-B resemble GRK4 and GRK5, respectively, in terms of the structural and functional properties of their C-termini leads us to suggest that GRK6-C may be considered a basic, prototypic representative of the GRK4-like kinases, which is capable of interacting with both plasma membrane and its receptor substrate, but is resistant to further regulatory modification conferred to the prototype via C-terminal extension The fact that all three GRK6 isoforms, in contrast to the isoforms of GRK4 [8], are conserved between several mammalian species indicates that the existence of functionally distinct forms of GRK6 is biologically important The cDNAs of the coding regions of mGRK6-A, mGRK6C and mGRK6-D were amplified by PCR from singlestranded cDNA prepared from mouse mesenterial lymph nodes as described previously [7] Complementary DNAs encoding C-terminal mGRK6-A mutants were prepared from the wild-type mGRK6-A cDNA by PCR In brief, mGRK6-A mutant M1 was generated according to the protocol of the QuickChangetm site-directed mutagenesis kit (Stratagene, La Jolla, CA) (18 cycles: 94 °C for min, 55 °C for min, 72 °C for min, followed by a single incubation at 72 °C for 10 min) using primer P1, 5¢-CGCCA AGATTCCTCTGGGAACTCCAGCGACAGT-3¢ (nucleotides 1677–1709, sense) in combination with primer P2, 5¢-ACTGTCGCTGGAGTTCCCAGAGGAATCTTGG CG-3¢ (nucleotides 1677–1709, antisense) Complementary DNAs encoding the C-terminal mGRK6-A mutants M2 and M3 were prepared either from the mGRK6-A mutant M1 or from the wild-type mGRK6-A cDNA by PCR (30 cycles: 94 °C for min, 70 °C or 62 for min, 72 °C for min, followed by a single incubation at 72 °C for 10 min) using primer P1, 5¢-AGCCCATGGAGCTCGAGAACA TCGTA-3¢ (nucleotides 1–26, sense, initiating ATG underlined) in combination with antisense primers introducing a stop codon sequence: mGRK6-A M2: P2, 5¢-CTAGTCGC TGGAGTTCCCAGAGGAATCTTGGCG-3¢ (nucleotides 1677–1706, antisense, stop codon underlined) and mGRK6A M3: P3, 5¢-CTAATCTTGGCGACTGAAGAGTCT-3¢ (nucleotides 1665–1685, antisense, stop codon underlined) The PCR products were ligated into pCR 2.1 (Invitrogen, Carlsbad, CA) and their identities were verified by DNA sequencing The numbering of oligonucleotides used as primers refers to the nucleotide sequence of the mGRK6-A cDNA deposited with EMBL ⁄ GenBank under Accession no Y15799 Experimental procedures Production of recombinant baculoviruses Materials PtdCho (P-7763), PtdSer (P-7769), PtdIns (P-8443), PtdInsP (P-9638) and PtdInsP2 (P-9763) were obtained from Sigma (Taufkirchen, Germany) Antisera PV1 and PV2 were raised in rabbits against synthetic peptides corresponding to residues 87–101 (H2N-V87SEYEVTPDEKRKAC-CONH2) and 574–589 (H2N-CP574PASSPQAEAPTGGWR-COOH), respectively, of mGRK6-B An N-terminal cysteine was added to the latter peptide to facilitate coupling to the carrier protein The rabbit polyclonal antiserum sc-566 reactive against amino acids 557–576 of mGRK6-A was purchased from Santa Cruz Biotechnology (Santa Cruz, CA) Triton X-100 (PT-8787) was obtained from Sigma Construction of wild-type and mutant GRK6 cDNAs The cDNAs of wild-type and mutant mGRK6 isoforms were prepared by endonuclease digestion from pCR 2.1, filled in with Klenow enzyme, and subcloned into the SmaI site of the baculovirus transfer vector pVL1393 (Invitrogen) The correct orientation of the inserts was verified by DNA sequencing Recombinant baculoviruses were obtained by transfecting Sf9 cells with a 25 : (v ⁄ v) mixture of the transfer vector and a modified baculovirus FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS 6047 Variable C-terminus of GRK6 mediates autoregulation P Vatter et al DNA (Baculogold; BD Biosciences-Pharmigen, Franklin Lakes, NJ, USA), which contains a lethal deletion and is rescued by the DNA of the transfer vector High-titre stocks of the baculoviruses were produced by three cycles of amplification in Sf9 cells Production of recombinant mGRK6 proteins in baculovirus-infected insect cells Sf9 cells were grown at 27 °C in TNM-FH medium (Sigma) supplemented with 50 lgỈmL)1 gentamycin and 10% (v ⁄ v) fetal bovine serum in 75-cm2 cell culture flasks Cells (1 · 107 cells per flask) were incubated for 48 h with recombinant baculovirus in 15 mL medium Two days after infection, cells were collected by centrifugation, rinsed once with NaCl ⁄ Pi (140 mm NaCl, 2.7 mm KCl, 8.0 mm Na2HPO4, 1.4 mm KH2PO4, pH 7.2) and resuspended in 200 lL icecold lysis buffer A (20 mm Hepes ⁄ NaOH, pH 7.5, 250 mm NaCl, 10 mm EDTA, mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 200 lgỈmL)1 benzamidine, and 20 lgỈmL)1 leupeptin) Cells were homogenized by forcing the suspension six times through a 0.5 · 23 mm needle attached to a disposable syringe The lysate was centrifuged at 40 000 g for 30 at °C, and the supernatant was centrifuged again at 300 000 g for 30 at °C and then passed through 0.22 lm pore size nitrocellulose filters To produce larger quantities of recombinant mGRK6 proteins, Sf9 cells were grown at 27 °C in suspension culture in Grace’s Complete Insect Medium (BioWhittaker) supplemented with 0.2% (w ⁄ v) Pluronic F-68 (Gibco BRL, Rockville, MD), 50 lgỈmL)1 gentamycin, and 2.5 lgỈmL)1 amphotericin B (Fungizone, Gibco BRL) Cells (8 · 108 per flask) were incubated for 48 h with recombinant baculovirus in 400 mL medium at 80 r.p.m on a rotary shaker with amplitude of 25 mm The cells were collected by centrifugation, washed once in ice-cold NaCl ⁄ Pi, resuspended in 40 mL lysis buffer A and then homogenized and fractionated as described above The mGRK6 proteins mGRK6-A through -C were similarly abundant in soluble fractions of baculovirus-infected insect cells ( 1% of total protein) The catalytically inactive variant mGRK6-D was at least 10-fold less abundant in this fraction (Fig 1B, left) Purification of recombinant mGRK6-C Recombinant mGRK6-C was purified from the soluble fraction of baculovirus-infected insect cells by sequential chromatography on SP Sepharose high performance and heparin ă Sepharose high performance using an AKTAexplorer chromatography system (Amersham Biosciences, Freiburg, Germany) The soluble fraction from Sf9 cells infected with baculovirus encoding mGRK6 (40 mL, 260 mg protein) was diluted with 80 mL of ice-cold buffer B (20 mm Hepes ⁄ NaOH, pH 7.5, 10 mm EDTA, mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 200 lgỈmL)1 benz- 6048 amidine and 20 lgỈmL)1 leupeptin) and applied to a mL HiTrap SP HP column (Amersham Biosciences) that had been equilibrated with buffer B containing 125 mm NaCl The flow rate was mLỈmin)1 After application of the sample, the column was washed with 40 mL of buffer B containing 125 mm NaCl and eluted with a linear gradient (50 mL) of NaCl (125–500 mm) in buffer B Fractions of mL were collected and analysed by SDS ⁄ PAGE and immunoblotting using antiserum PV1, and by measuring the ability of the fractions to phosphorylate light-activated rhodopsin The active material, which eluted at  350 mm NaCl, was pooled (10 mL, 20 mg of protein) and diluted with buffer B to obtain a final NaCl concentration of 300 mm The sample was then applied to a mL HiTrap Heparin HP column (Amersham Biosciences), which had been equilibrated with buffer B containing 300 mm NaCl The flow rate was 0.5 mLỈmin)1 After application of the sample, the column was washed with 30 mL of buffer B containing 300 mm NaCl and eluted with a linear gradient (40 mL) of NaCl (0.3–1 m) in buffer A Fractions of mL were collected and analysed by SDS ⁄ PAGE and staining of proteins with silver The active material eluted at  600 mm NaCl and was directly used in mGRK6 phospholipid interaction studies Expression of recombinant mGRK6 proteins in COS-7 cells The cDNAs encoding mGRK6-A to -D were ligated into the mammalian expression vector pMT2 [41] COS-7 cells were grown in six well dishes (35 mm wells, · 105 cells per well) in Dulbecco’s modified Eagle’s medium supplemented with 10% (v ⁄ v) fetal bovine serum, mm l-glutamine, 100 unitsỈmL)1 penicillin, 0.1 mgỈmL)1 streptomycin, mm sodium pyruvate and 25 mm Hepes ⁄ NaOH, pH 7.2 at 37 °C, in a humidified atmosphere of 90% air and 10% CO2 Cells were transfected by lipofection (2 lg plasmid DNA per well) using SuperFecttm (Qiagen, Hilden, Germany) according the manufacturer’s instructions For immunoblot analysis, transfected COS-7 cells were harvested by scraping into mL of ice-cold NaCl ⁄ Pi 48 h after transfection Cells were sedimented by centrifugation and resuspended in 200 lL ice-cold lysis buffer A (20 mm Tris ⁄ HCl, pH 7.5, mm EDTA pH 7.5, 0.5 mm phenylmethylsulfonyl fluoride, 20 lgỈmL)1 aprotinin and 10 lgỈmL)1 leupeptin) and homogenized by forcing the suspension six times through a 0.5 · 23 mm needle attached to a disposable syringe The lysate was centrifuged at 300 g for at °C, and the supernatant was centrifuged again at 300 000 g for 30 at °C The supernatant was removed and the particulate fraction was resuspended in 200 lL of lysis buffer A To solubilize proteins from 150 lL of the membrane fraction, 50 lL of a solution containing 400 mm NaCl, mm EDTA and 6% (v ⁄ v) Triton X-100 was added The mixture was incubated for h at °C by end-over-end rotation and then centrifuged FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS P Vatter et al at 300 000 g for 30 at °C The supernatant was recovered, snap-frozen in liquid nitrogen and stored at )80 °C The three catalytically active mGRK6 variants mGRK6-A to -C were present at similar levels in soluble, particulate, and detergent-solubilized particulate fractions of COS-7 cells that had been transfected with identical amounts of vector DNA (Fig 2A) Mouse mGRK6-D was only found and was present at substantially lower levels in the particulate fraction upon subcellular fractionation and was resistant to detergent solubilization (not shown) Immunocytochemistry and confocal microscopy of transfected COS-7 cells Eight hours after transfection, cells were trypsinized, plated onto 12-mm glass coverslips, and cultured for 40 h in a 24-well plate Unless specified otherwise, the following steps were carried out at room temperature Forty-eight hours after transfection, the cells were rinsed twice with NaCl ⁄ Pi and fixed for 30 with NaCl ⁄ Pi containing 4% (w ⁄ v) paraformaldehyde Fixed cells were rinsed three times with NaCl ⁄ Pi and permeabilized for 10 with NaCl ⁄ Pi containing 0.2% (v ⁄ v) Triton X-100 The cells were blocked for 30 at 37 °C with NaCl ⁄ Pi containing 0.05% (v ⁄ v) Triton X-100 (PBST) and 5% (w ⁄ v) nonfat dried milk and subsequently incubated for h at 37 °C with antiserum PV1 diluted : 200 in PBST The cells were then rinsed three times with PBST and incubated for h at 37 °C with a Cytm 3-conjugated goat anti-rabbit serum (Dianova, Hamburg, Germany) diluted : 500 in the same buffer Cells were rinsed five times with PBST, once with NaCl ⁄ Pi, once with double-distilled water and the coverslips were mounted on a slide Fluorescence microscopy was performed on a TCS 4D confocal microscope (Leica, Wetzlar, Germany) using a ·60 objective with oil immersion Interaction of recombinant mGRK6-C with phospholipid vesicles The phospholipids were diluted in chloroform, mixed and evaporated to dryness in a 14 · 100 mm Pyrex tube under a stream of nitrogen at °C The lipids were then resuspended by vortex mixing for 15 at room temperature in vesicle buffer (50 lL, 17 mg phospholipidỈ mL)1) containing 10 mm Tris ⁄ HCl, pH 8.0, 100 mm NaCl and 0.1 mm EDTA The suspension was then sonicated for 15 at °C in a bath type sonicator (Sonorex RK 102; Bandelin, Berlin, Germany) Aliquots (3 lL) of the freshly prepared phospholipid vesicle preparation were incubated for 10 at °C in a final volume of 30 lL containing 20 mm Tris ⁄ HCl, pH 8.0, 100 mm NaCl, mm MgCl2 and lg of purified mGRK6-C Incubation mixtures were separated into soluble and particulate constituents by centrifugation at 100 000 g for 25 at °C Variable C-terminus of GRK6 mediates autoregulation The soluble supernatant was removed and the membrane fraction was resuspended in 30 lL of incubation buffer Aliquots (15 lL) of the supernatant and resuspended membrane fraction were analysed by immunoblotting Phosphorylation of light-activated rhodopsin Retinal rod outer segment membranes were prepared at °C under dim red light from bovine eyes as described by Papermaster [42] Rhodopsin kinase-free rod outer segment membranes were prepared by treating the membranes with buffer containing 50 mm Tris ⁄ HCl, pH 8.0, mm EDTA and m urea as described by Shichi and Somers [43] Urea-treated rod outer segment membranes consisted to  90% of rhodopsin, as assessed by SDS ⁄ PAGE, and showed negligible endogenous rhodopsin kinase activity, as determined by analysing the lightinduced phosphorylation of rhodopsin Phosphorylation of rhodopsin was assayed in a reaction mixture (15 lL) containing rod outer segment membranes (80 ng protein) and soluble wild-type or mutant mGRK6 (5 lg protein) in buffer containing 20 mm Tris ⁄ HCl, pH 7.5, mm EDTA, 3.7 mm MgCl2 and 100 lm [32P]ATP[cP] (specific activity CiỈmmol)1) Incubations were carried out for 10 at 30 °C in ambient light, and were stopped by the addition of 15 lL of SDS sample buffer [125 mm Tris ⁄ HCl, pH 6.8, 4% (w ⁄ v) SDS, 20% (v ⁄ v) glycerol, 10% (v ⁄ v) b-mercaptoethanol, and 0.002% (w ⁄ v) pyronin Y] Radiolabeled proteins were visualized by SDS ⁄ PAGE and autoradiography of the gel Miscellaneous Protein concentrations were determined according to Bradford [44] using bovine IgG as standard SDS ⁄ PAGE and immunoblotting were performed according to standard protocols [45], except that immunoreactive proteins were visualized using the ECL western blotting detection system (Amersham Biosciences) The method used to stain polyacrylamide gels with silver is specified in Oakley et al [46] All cloned PCR products were sequenced in both directions by primer walking using the ABI PrismÒ BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit on an ABI PRISMTM 310 sequencer (Applied Biosystems, Darmstadt, Germany) No differences between the actual and the expected sequences were detected Acknowledgements This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 451) The expert technical assistance of Stephan Rutten, Ingrid Busselă ă mann and Susanne Gierschik is greatly appreciated FEBS Journal 272 (2005) 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expression in mammalian cells Methods Enzymol 185, 487–511 42 Shichi H & Somers RL (1978) Light-dependent phosphorylation of rhodopsin Purification and properties of rhodopsin kinase J Biol Chem 253, 7040–7046 43 Papermaster DS (1982) Preparation of retinal rod outer segments Methods Enzymol 81, 48–52 44 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254 45 Harlow E & Lane D (1988) Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 46 Oakley BR, Kirsch DR & Morris NR (1980) A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels Anal Biochem 105, 361–363 FEBS Journal 272 (2005) 6039–6051 ª 2005 The Authors Journal compilation ª 2005 FEBS 6051 ... not only enhance the hydrophobicity and thereby strengthen the membrane association of GRK6-A, but also increase the kinase catalytic activity of the protein Along the same lines, the C-terminal. .. removal of the C-terminal- most 16 residues of mGRK6-A M1 affected the activity of the protein toward rhodopsin in a nonuniform manner Thus, removal of the C-terminal- most nine residues of mGRK6-A... the activities of the 60 44 C-terminally extended variants mGRK6-A and -B (Fig 6) These results suggested that the C-terminal extensions present in the latter two isoforms may reduce their ability